U.S. patent number 9,315,828 [Application Number 12/532,949] was granted by the patent office on 2016-04-19 for prompt nucleic acid delivery carrier composition.
This patent grant is currently assigned to OTSUKA PHARMACEUTICAL CO., LTD., Hirofumi Takeuchi. The grantee listed for this patent is Yasuyuki Hira, Koji Nakano, Hirofumi Takeuchi, Hidekazu Toyobuku. Invention is credited to Yasuyuki Hira, Koji Nakano, Hirofumi Takeuchi, Hidekazu Toyobuku.
United States Patent |
9,315,828 |
Takeuchi , et al. |
April 19, 2016 |
Prompt nucleic acid delivery carrier composition
Abstract
An object of the present invention is to provide a carrier
composition for nucleic acid delivery, which can efficiently
deliver a nucleic acid into cells when a nucleic acid such as siRNA
is administered to animal-derived cells or animals, and also has
low toxicity and high safety, and a composition for nucleic acid
delivery containing the carrier composition and nucleic acid. A
carrier for nucleic acid delivery is prepared by using (A) a
diacylphosphatidylcholine, (B) at least one member selected from
the group consisting of cholesterol and derivatives thereof, and
(C) an aliphatic primary amine. Also, a composition for nucleic
acid delivery is prepared by mixing the carrier for nucleic acid
delivery with a nucleic acid.
Inventors: |
Takeuchi; Hirofumi (Gifu,
JP), Hira; Yasuyuki (Gifu, JP), Nakano;
Koji (Gifu, JP), Toyobuku; Hidekazu (Tokushima,
JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Takeuchi; Hirofumi
Hira; Yasuyuki
Nakano; Koji
Toyobuku; Hidekazu |
Gifu
Gifu
Gifu
Tokushima |
N/A
N/A
N/A
N/A |
JP
JP
JP
JP |
|
|
Assignee: |
Takeuchi; Hirofumi (Gifu,
JP)
OTSUKA PHARMACEUTICAL CO., LTD. (Tokyo, JP)
|
Family
ID: |
39788561 |
Appl.
No.: |
12/532,949 |
Filed: |
March 26, 2008 |
PCT
Filed: |
March 26, 2008 |
PCT No.: |
PCT/JP2008/055730 |
371(c)(1),(2),(4) Date: |
September 24, 2009 |
PCT
Pub. No.: |
WO2008/117828 |
PCT
Pub. Date: |
October 02, 2008 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20100063131 A1 |
Mar 11, 2010 |
|
Foreign Application Priority Data
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|
|
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Mar 26, 2007 [JP] |
|
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2007-79944 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/713 (20130101); C12N 15/111 (20130101); C12N
15/87 (20130101); A61K 31/7088 (20130101); A61P
37/00 (20180101); A61K 9/127 (20130101); A61P
35/00 (20180101); A61K 48/00 (20130101); C12N
2310/14 (20130101); C12N 2320/32 (20130101) |
Current International
Class: |
A61K
48/00 (20060101); C07H 21/02 (20060101); A61K
9/127 (20060101); C12N 15/87 (20060101); C12N
15/88 (20060101); A61K 31/7088 (20060101); A61K
31/713 (20060101); C12N 15/11 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
1759691 |
|
Mar 2007 |
|
EP |
|
10-313872 |
|
Dec 1998 |
|
JP |
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11-292795 |
|
Oct 1999 |
|
JP |
|
2002-529439 |
|
Sep 2000 |
|
JP |
|
2005-508394 |
|
Mar 2005 |
|
JP |
|
2005-336081 |
|
Dec 2005 |
|
JP |
|
95/24201 |
|
Sep 1995 |
|
WO |
|
99/13816 |
|
Mar 1999 |
|
WO |
|
2004/017940 |
|
Mar 2004 |
|
WO |
|
2005/007196 |
|
Jan 2005 |
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WO |
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2005/102268 |
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Nov 2005 |
|
WO |
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2006/002538 |
|
Jan 2006 |
|
WO |
|
WO 2007/048019 |
|
Apr 2007 |
|
WO |
|
2009/061003 |
|
May 2009 |
|
WO |
|
Other References
Takano et al (Pharmaceutical Research, vol. 20, No. 7, Jul. 2003).
cited by examiner .
Product information for L-alpha-phosphatidylcholine from Sigma
Chemical Company, retrieved from
http://www.sigmaaldrich.com/catalog/product/sigma/61755?lang=en®ion=US
on Oct. 15, 2013. cited by examiner .
International Search Report mailed May 27, 2008 in
PCT/JP2008/055730. cited by applicant .
Extended European Search Report issued in corresponding EP
Application No. 08738918.5 on Aug. 9, 2011 (in the name of Otsuka
Pharmaceutical Co., Ltd. et al.). cited by applicant .
Maria Kokkona et al., "Stability of SUV liposomes in the presence
of cholate salts and pancreatic. ipases: effect of lipid
composition", European Journal of Pharmaceutical Sciences, 2000, 9:
245-252. cited by applicant .
Yoshie Maitani et al., "Physicochemical Characteristics and
Transfection Efficienty of DNA in Liposomes with Soybean-Derived
Sterylglucoside into HepG2 Cells", J. Pharm. Sci. Technol., Japan,
2001, 61(1): 1-10. cited by applicant .
Office Action issued Apr. 8, 2013, in corresponding European Patent
Application No. 08738918.5. cited by applicant.
|
Primary Examiner: Schnizer; Richard
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
The invention claimed is:
1. A composition for delivery of siRNA comprising an siRNA, and a
carrier composition for delivery of siRNA, consisting essentially
of (A) at least one member selected from the group consisting of
dimyristoylphosphatidycholine and distearoylphosphatidylcholine ,
(B) cholesterol , and (C) an aliphatic primary amine, wherein the
molar ratio of component (A): component (B): component (C) is
6-9:1-4:1, wherein the total amount of components (A) to (C)
contained in the composition for delivery of siRNA is from 30 to
90% by weight, based on the total amount of the composition.
2. The composition for delivery of siRNA according to claim 1,
wherein the component (C) is an alkylamine having 10 to 20 carbon
atoms.
3. The composition for delivery of siRNA according to claim 1,
wherein the component (A) is at least one member selected from the
group consisting of dimyristoylphosphatidylcholine, and
distearoylphosphatidylcholine; the component (B) is cholesterol;
and the component (C) is stearylamine.
4. The composition for delivery of siRNA according to claim 1,
wherein the carrier composition for delivery of siRNA is a liposome
preparation in which a liposomal membrane is formed of the
components (A) to (C).
5. The composition for delivery of siRNA according to claim 1,
wherein the composition is a liposomal preparation.
6. A method for introducing a siRNA, which comprises the step of
introducing the siRNA into cells by bringing the composition for of
siRNA delivery of claim 1 into contact with the cells.
7. A method for introducing a siRNA according to claim 6, wherein
the cells are cultured cells, cells separated from living
organisms, or cells existing in living organisms.
Description
TECHNICAL FIELD
The present invention relates to a carrier composition for nucleic
acid delivery which can efficiently deliver a nucleic acid into
cells when a nucleic acid is administrated to animal-derived cells
or organisms, and which also has low toxicity and high safety; and
to a composition for nucleic acid delivery.
BACKGROUND ART
Various types of nucleic acids which exert physiologically active
functions within cells have been discovered through recent
developments in biotechnology. For example, it is known that small
interfering RNA (siRNA) induces degradation of mRNA of a target
gene existing within cells and inhibits expression of the target
gene (RNA interference). The inhibitory function against target
gene expression due to the RNA interference is useful for
mitigation or treatment of disease symptoms caused by irregular
expression of particular genes or gene groups, and development of
therapeutic agents using siRNA is expected. To utilize nucleic
acids including siRNA as therapeutic agents, it is important that
siRNA functions in the target cell, and therefore, it is essential
to establish efficient techniques to deliver the nucleic acids into
target cells.
Use of a carrier (vector) is known as a technique to deliver
exogenous nucleic acid molecules or genes into cells. Vectors
include virus vectors and nonvirus vectors. Virus vectors have high
gene transfer efficiency; however, there are various unknown safety
aspects including pathogenicity, immunogenicity and cytotoxicity.
Therefore, development of safer nonvirus vectors is awaited.
As a nonvirus nucleic acid delivery carrier that promotes delivery
of a nucleic acid, such as siRNA, into cells, for example, a
cationic lipid with a specific structure has been reported in
Patent Document 1. However, the cationic lipid reported in Patent
Document 1 has a disadvantage that it shows toxicity when
administered to cultured cells or living organisms. Also, Patent
Document 2 discloses a composition containing an amphiphilic
compound and a polycation as a carrier composition which has
comparatively low toxicity and can deliver siRNA into cells.
However, the composition reported in Patent Document 2 also has a
safety problem since its cytotoxicity becomes non-negligible when a
sufficient amount of siRNA is introduced into cells.
In light of the prior art, development of a carrier composition for
nucleic acid delivery which has low toxicity and can efficiently
deliver a nucleic acid, such as siRNA, into cells has been desired.
[Patent Document 1] Japanese Unexamined Patent Publication No.
2002-529439 [Patent Document 2] Japanese Unexamined Patent
Publication No. 2005-508394
DISCLOSURE OF THE INVENTION
Technical Problem
Thus, an object of the present invention is to solve the above
problems of the prior art. Specifically, an object of the present
invention is to provide a carrier composition for nucleic acid
delivery, which can efficiently deliver nucleic acid into cells
when a nucleic acid such as siRNA is administered to animal-derived
cells or animals, and also has low toxicity and high safety; and a
composition for nucleic acid delivery containing the carrier
composition and nucleic acid. Also, an another object of the
present invention is to provide a method for introducing a nucleic
acid into cells, which can efficiently deliver the nucleic acid
into cells with high safety.
Means for Solving the Problem
The present inventors have intensively studied so as to achieve the
above object and found that a composition containing (A) a
diacylphosphatidylcholine, (B) cholesterol and/or a derivative
thereof and (C) an aliphatic primary amine has low toxicity and
high safety, can efficiently deliver a nucleic acid into cells, and
is therefore useful as a carrier for nucleic acid delivery. They
have also found that it is possible to impart more excellent safety
and nucleic acid-introducing properties by using a composition
containing the components (A) to (C) after forming into a liposomal
form. The present invention has been completed by making further
improvement based on these findings.
Namely, the present invention provides the following
embodiments.
Item 1. A carrier composition for nucleic acid delivery, comprising
(A) a diacylphosphatidylcholine, (B) at least one member selected
from the group consisting of cholesterol and derivatives thereof,
and (C) an aliphatic primary amine.
Item 2. The carrier composition for nucleic acid delivery according
to Item 1, wherein the component (A) is a diacylphosphatidylcholine
whose acyl group moiety has 4 to 23 carbon atoms.
Item 3. The carrier composition for nucleic acid delivery according
to Item 1 or 2, wherein the component (B) is cholesterol.
Item 4. The carrier composition for nucleic acid delivery according
to Item 1, wherein the component (C) is an alkylamine having 10 to
20 carbon atoms.
Item 5. The carrier composition for nucleic acid delivery according
to Item 1,
wherein the component (A) is at least one member selecting from the
group consisting of dimyristoylphosphatidylcholine,
dipalmitoylphosphatidylcholine, and
distearoylphosphatidylcholine;
the component (B) is cholesterol; and
the component (C) is stearylamine.
Item 6. The carrier composition for nucleic acid delivery according
to Item 1, wherein the molar ratio of component (A):component
(B):component (C) is 5-9:1-5:1.
Item 7. The carrier composition for nucleic acid delivery according
to Item 1, which is a carrier for delivery of siRNA.
Item 8. The carrier composition for nucleic acid delivery according
to Item 1, which is a liposome preparation in which a liposomal
membrane is formed of the components (A) to (C).
Item 9. A composition for nucleic acid delivery, comprising a
nucleic acid, and the carrier composition for nucleic acid delivery
of Item 1.
Item 10. The composition for nucleic acid delivery according to
Item 9, wherein the nucleic acid is siRNA.
Item 11. The composition for nucleic acid delivery according to
Item 9, which is a liposome preparation.
Item 12. A method for introducing a nucleic acid, which comprises
the step of introducing the nucleic acid into cells by bringing the
composition for nucleic acid delivery of Item 9 into contact with
the cells.
Item 13. A method for introducing a nucleic acid according to Item
12, wherein the cells are cultured cells, cells separated from
living organisms, or cells existing in living organisms.
Item 14. Use of a composition comprising (A) a
diacylphosphatidylcholine, (B) at least one member selected from
the group consisting of cholesterol and derivatives thereof, and
(C) an aliphatic primary amine, in the manufacture of a carrier for
nucleic acid delivery.
Item 15. Use according to Item 14, wherein the carrier is used for
delivery of siRNA.
EFFECTS OF THE INVENTION
The carrier composition for nucleic acid delivery and the
composition for nucleic acid delivery of the invention have an
advantage that they can effectively deliver a nucleic acid into
cells thereby exerting a useful function of the nucleic acid in
cells, and also has low toxicity and high safety. Therefore, the
carrier composition for nucleic acid delivery and the composition
for nucleic acid delivery are useful for treatment of various
diseases by introduction of a nucleic acid, particularly treatment
of intractable diseases which are difficult to be treated by a low
molecular compound.
The carrier composition for nucleic acid delivery and the
composition for nucleic acid delivery of the invention are
particularly preferred to introduce siRNA into cells since
induction of interferon expression, which is an adverse reaction of
siRNA, can be effectively suppressed.
Furthermore, the composition for nucleic acid delivery of the
invention also has an advantage that the composition can be stored
in a freeze-dried state since it can be subjected to a
freeze-drying treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of Test Example 1, i.e., the results of
the evaluation of a composition for nucleic acid delivery on safety
for cells.
FIG. 2 shows the results of the evaluation of siRNA introduction
into cells mediated by each composition for nucleic acid delivery
in Test Example 2. The ordinate in FIG. 2 indicates the average
fluorescence intensity per one cell.
FIG. 3 shows the results of the evaluation of siRNA introduction
into cells when the concentration of the constituent lipids (DSPC,
cholesterol and stearylamine) of the carrier for nucleic acid
delivery is changed in a composition for nucleic acid delivery in
Test Example 2.
FIG. 4 shows the evaluation results of the inhibition of interferon
induction by a siRNA-containing composition for nucleic acid
delivery in Test Example 3.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will now be described in detail.
Carrier for Nucleic Acid Delivery
The carrier composition for nucleic acid delivery of the invention
comprises (A) a diacylphosphatidylcholine, (B) cholesterol and/or a
derivative thereof, and (C) an aliphatic primary amine.
The carrier composition for nucleic acid delivery of the invention
is used as a nucleic acid carrier for delivery (introduction) of a
nucleic acid into cells.
The type and structure of the nucleic acid to which the carrier
composition for nucleic acid delivery of the present invention is
applied is not limited as long as it is required to be delivered
into cells. Specific examples of such nucleic acids include siRNA,
mRNA, tRNA, rRNA, cDNA, miRNA (microRNA), ribozyme, antisense
oligodeoxynucleotide, decoy oligonucleotide, plasmid DNA, peptide
nucleic acid, triplex forming oligonucleotide (TFO), aptamer, and
genes. Particularly, the carrier composition for nucleic acid
delivery of the invention has the useful feature of inhibiting
induction of interferon expression, which is an adverse reaction of
siRNA, and thus it is useful to deliver siRNA into cells. Nucleic
acids to which the carrier composition for nucleic acid delivery of
the invention is applied may be derived from humans, animals,
plants, bacteria and viruses, and also, it may be produced by
chemical synthesis. In addition, these nucleic acids can be single,
double or triple strand, and the molecular weight thereof is not
specifically limited. Also, nucleic acids can be modified with
chemical compounds, enzymes or peptides. In the invention, these
nucleic acids may be used alone, or two or more kinds of them may
be used in combination.
Diacylphosphatidylcholine (hereinafter, referred sometimes to as
"component (A)") used in the carrier composition for nucleic acid
delivery of the invention is not specifically limited as long as it
is pharmacologically acceptable, and examples thereof include a
diacylphosphatidylcholine whose acyl group moiety has 4 to 23
carbon atoms. The number of carbon atoms of two acyl groups
constituting the diacylphosphatidylcholine may be the same or
different.
Specific examples of the diacylphosphatidylcholine used in the
present invention include dilauroylphosphatidylcholine,
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, dioleoylphosphatidylcholine,
dilinoleoylphosphatidylcholine,
myristoylpalmitoylphosphatidylcholine,
myristoylstearoylphosphatidylcholine,
palmitoylstearoylphosphatidylcholine,
dibutyloylphosphatidylcholine, dihexanoylphosphatidylcholine,
diheptanoylphosphatidylcholine, didecanoylphosphatidylcholine,
diphthanoylphosphatidylcholine, didodecylphosphatidylcholine,
dieicosenoylphosphatidylcholine, dihenicosanoylphosphatidylcholine,
dierucoylphosphatidylcholine, diarachidonoylphosphatidylcholine,
and bis(tricosadinoyl)phosphatidylcholine. Of these
diacylphosphatidylcholines, a diacylphosphatidylcholine whose acyl
group moiety has 12 to 18 carbon atoms is preferable; a
diacylphosphatidylcholine whose acyl group moiety has 13 to 17
carbon atoms, such as dimyristoylphosphatidylcholine,
dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,
myristoylpalmitoylphosphatidylcholine,
myristoylstearoylphosphatidylcholine, and
palmitoylstearoylphosphatidylcholine is more preferable;
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and
distearoylphosphatidylcholine are particularly preferable; and
distearoylphosphatidylcholine is most preferable. These
diacylphosphatidylcholines may be used alone, or two or more kinds
of them may be used in combination.
Cholesterol and/or a derivative thereof (hereinafter referred
sometimes to as "component (B)") used in the carrier composition
for nucleic acid delivery of the present invention is not
specifically limited as long as it is pharmacologically acceptable.
The derivative of cholesterol is a cationic lipid having a
cholesterol skeleton, and specific examples thereof include
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
(DC-Chol), 3.beta.-[N',N',N'-trimethylaminoethane]cholesterol
iodide (TC-Chol), bis(guanidium)-tren-cholesterose (BGTC),
N-cholesteryloxycarbonyl-3,7-diazanonan-1,9-diamine,
.beta.-alanine-diethanolamine-cholesterol, N.sup.4-spermine
cholesteryl carbamate (GL-67) ,
N[N.sup.4-3-aminopropylspermidine]cholesteryl carbamate (GL-78) ,
N.sup.4-spermine cholesteryl carboxamide (GL-90) ,
N.sup.1,N.sup.8-bis(argininc carboxamide)-N.sup.4-spermidine
cholesteryl carbamate, and
N-[N.sup.1,N.sup.4,N.sup.8-tris(3-aminopropyl)spermidine]cholesteryl
carbamate (GL-96). In the invention, the component (B) is
preferably cholesterol. In the invention, as the component (B),
cholesterol and derivative thereofs may be used alone, or two or
more kinds of them may be used in combination.
The aliphatic primary amine (hereinafter referred sometimes to as
"component (C)") used in the carrier composition for nucleic acid
delivery of the invention is not specifically limited as long as it
is pharmacologically acceptable, and examples thereof include an
alkylamine whose alkyl group moiety has 10 to 20 carbon atoms.
Specific examples of the aliphatic primary amine used in the
present invention include laurylamine, myristylamine,
palmitylamine, stearylamine, oleylamine, decanoylamine, and
phthanoylamine. Of these aliphatic primary amines, an alkylamine
whose alkyl group moiety has 12 to 18 carbon atoms is preferable;
stearylamine, oleylamine and palmitoylamine are more preferable;
and stearylamine is particularly preferable. These dialiphatic
primary amines may be used alone, or two or more kinds of them may
be used in combination.
The carrier composition for nucleic acid delivery of the present
invention may contain a combination of the components (A) to (C).
In view of further enhancing efficiency of delivery of nucleic acid
into cells and low toxicity by employing the following
combinations, a combination of (A) a diacylphosphatidylcholine
whose acyl group moiety have 4 to 23 carbon atoms, (B) cholesterol
and/or a derivative thereof, and (C) an alkylamine having 10 to 20
carbon atoms is preferable and a combination of (A)
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine
and/or distearoylphosphatidylcholine, (B) cholesterol, and (C)
stearylamine is more preferable.
In the carrier composition for nucleic acid delivery of the present
invention, the ratio of the components (A) to (C) is not
specifically limited. For example, the molar ratio component
(A):component (B):component (C) is 5-9:1-5:1, preferably 6-9:1-4:1,
and more preferably 7-8:2-3:1. Efficiency of delivery of a nucleic
acid into cells and low toxicity can be further enhanced by
satisfying the molar ratio.
The total amount of the components (A) to (C) based on the total
amount of the carrier composition for nucleic acid delivery of the
present invention is, for example, from 1 to 100% by weight,
preferably from 20 to 90% by weight, and more preferably from 30 to
70% by weight.
The carrier composition for nucleic acid delivery of the present
invention may contain, in addition to the components (A) to (C),
other cationic lipids. Specific examples of the cationic lipid
include cationic lipids bonded with a steroid, such as squalamine,
3a,7a,12a-tris(3-aminopropoxy)-5.beta.-cholan-24-(N,N-bis(3-aminopropyl)a-
mine,
3a,7a,12a-tris(3-aminopropoxy)-5.beta.-cholan-24-(N--(N-(3-aminoprop-
yl))-3-aminopropyl)-amine),
3a,7a,12a-tris(3-azidopropoxy)-5.beta.-cholan-24-(N,N-bis(2-cyanoethyl)am-
ine), and
3a,7a,12a-tris(3-azidopropoxy)-5.beta.-cholan-24-(N-(benzyloxyca-
rbonyl)N-(3-hydroxypropyl)-amine)); cationic lipids bonded with
cholic acid, such as umbrella-spermine conjugates; cationic lipids
bonded with sterolglycoside; cationic lipids bonded with
steroidsaponin; and quaternary ammonium salt-type cationic lipids
such as dimethyldioctadecylammonium bromide (DDAB),
1,2-dimyristoyl-3-trimethylammoniumpropane,
1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP),
1,2-dioleoyl-3-trimethylammoniumpropanemethyl sulfate,
1,2-dipalmitoyl-3-trimethylammoniumpropane,
1,2-distearoyl-3-trimethylammoniumpropane,
N-(1-(2,3-bis(oleoyloxy)propyl)-N,N,N-trimethylammonium
hydrochloride (DOTMA),
dimyristoyloxypropyldimethylhydroxyethylammonium bromide (DMRIE),
dioleoyloxypropyldimethylhydroxyethylammonium bromide (DORIE),
dimethyldidodecylammonium bromide,
N-(a-trimethylammonioacetyl)-didodecyl-D-glutamine hydrochloride,
N-(a-trimethylammonioacetyl)-O,O'-bis-(1H,1H,2H,2H-perfluorodecyl-L-gluta-
mine hydrochloride,
O,O'-didodecanoyl-N-(a-trimethylammonioacetyl)diethanolamine
hydrochloride, methylallyldidodecylammonium bromide,
N-{p-(w-trimethylammoniobutyloxy)-benzoyl}-didodecyl-L-glutamine
hydrochloride,
9-(w-trimethylammoniobutyl)-3,6-bis(dodecanoyl)carbazole bromide,
dimethyldioctadecylammonium hydrochloride,
N-w-trimethylammoniodecanoyl-dihexadecyl-D-glutamine bromide,
N-{p-(w-trimethylammoniohexyloxy)-benzoyl}-ditetradecyl-L-glutamine
bromide, p-(w-trimethylammoniodecyloxy)-p'-octyloxyazobenzene
bromide (MC-1-0810),
p-{w-(b-hydroxyethyl)dimethyl-ammonio-decyloxy}-p'-octyloxyazobenzene
bromide (MC-3-0810) ,
O,O',O''-tridodecanoyl-N-(w-trimethyl-ammoniodecanoyl)-tris(hydroxymethyl-
)aminomethane bromide (TC-1-12) ,
1,2-dilauryl-glycero-3-ethylphosphocholine,
1,2-dimyristoyl-glycero-3-ethylphosphocholine,
1,2-dipalmitoyl-glycero-3-ethylphosphocholine,
1,2-distearoyl-glycero-3-ethylphosphocholine,
1,2-dioleoyl-glycero-3-ethylphosphocholine, and
1-palmitoyl-2-oleoyl-glycero-3-ethylphosphocholine.
In the invention, when cationic lipids other than the components
(A) to (C) are contained, the proportion of the cationic lipid is
not specifically limited as long as the effects of the invention
are not adversely affected. The proportion of the cationic lipid is
from 1 to 10 parts by weight, preferably from 2 to 8 parts by
weight, and more preferably from 4 to 6 parts by weight, per 100
parts by weight of the total amount of the components (A) to
(C).
Furthermore, the carrier composition for nucleic acid delivery of
the invention may contain an oily base, if necessary. By including
an oily base and using its characteristics, it becomes possible to
control the efficiency of the nucleic acid to be introduced by the
carrier composition for delivery. For example, adjustment of the
specific gravity of the carrier composition for nucleic acid
delivery by including the oily base controls contact between the
nucleic acid and the carrier composition for nucleic acid delivery
and allows improvement of introduction efficiency in vitro. In
addition, for example, by including a compound with a
temperature-sensitive function as the oily base, fluctuation on a
cell surface can be induced due to core disruption of a nucleic
acid carrier composition under a given temperature condition,
thereby improvement of introduction efficiency of nucleic acid
become possible. Furthermore, for example, by including a compound
with an external stimulation disruption property as the oily base,
fluctuation on the cell surface can be induced due to core
disruption of a nucleic acid carrier composition caused by external
stimulation, thereby improvement of introduction efficiency of the
nucleic acid become possible.
Examples of the oily base included in the carrier composition for
nucleic acid delivery of the present invention include
perfluorocarbon, perfluoropentane, perfluorooctyl bromide,
perfluorohexane, perfluorotributylamine, soybean oil, refined
soybean oil, hardened soybean oil, unsaponified soybean oil,
squalene, castor oil, clove oil, sorbitan trioleate, turpentine
oil, safflower oil, safflower oil fatty acid, oleic acid, coconut
oil, rapeseed oil, fusel oil, olive oil, linseed oil, sesame oil,
chlorophyll oil, croton oil, bergamot oil, cedar oil, orange oil,
fennel oil, eucalyptus oil, corn oil, lavender oil, sweet majoram
oil, lemon oil, cotton seed oil, egg york oil, rose oil, pine oil,
almond oil, peanut oil, camellia oil, white camphor oil, chamomile
oil, cinnamon oil, peppermint oil, esterified corn oil, bread oil,
Anthemis nobilis oil, snake oil, spearmint oil, sunflower oil,
cacao butter, wheat germ oil, zinc oxide oil, hardened oil,
hydrogenated vegetable oil, light liquid paraffin, liquid paraffin,
medium chain fatty acid triglyceride, mink oil, orange peel oil,
polyoxyethylene castor oil, polyoxyethylene hardened castor oil,
polyoxyethylene hardened castor oil 10, polyoxyethylene hardened
castor oil 100, polyoxyethylene hardened castor oil 20,
polyoxyethylene hardened castor oil 40, polyoxyethylene hardened
castor oil 5, polyoxyethylene hardened castor oil 50,
polyoxyethylene hardened castor oil 60, polyoxyl 35 castor oil, and
process oil. Of these oily bases, perfluoropentane has temperature
sensitivity and also has characteristics that it is volatilized at
29.5.degree. C. Also, perfluorohexane, perfluorooctyl bromide and
perfluorotributylamine have an external stimulation disruption
property and have characteristics that cavitation is generated on
the core of the carrier composition through external stimulation
such as stimulation caused by irradiation with ultrasound, thereby
causing core disruption.
When the carrier composition for nucleic acid delivery contains the
oily base, the proportion of the oily base is not specifically
limited as long as the effects of the invention are not adversely
affected. The proportion of the oily base is from 0.1 to 50 parts
by weight, preferably from 1 to 30 parts by weight, and more
preferably from 5 to 20 parts by weight, per 100 parts by weight of
the total amount of the components (A) to (C).
Furthermore, the carrier composition for nucleic acid delivery of
the invention may contain a membrane-fusogenic lipid (helper
lipid), if necessary. It becomes possible to further enhance
efficiency of delivery of the nucleic acid into cells by containing
such membrane-fusogenic lipid. Examples of the membrane-fusogenic
lipid include dioleoylphosphatidylethanolamine,
dioleoylphosphatidylcholine,
transphosphatidylphosphatidylethanolamine,
1,2-bis(10,12-tricosadinoyl)-phosphoethanolamine,
1,2-dielaidoylphosphoethanolamine,
1,2-dihexadecylphosphoethanolamine,
1,2-dihexanoylphosphoethanolamine,
1,2-dilauroylphosphoethanolamine,
1,2-dilinoleoylphosphoethanolamine,
1,2-dimyristoylphosphoethanolamine,
1,2-dioleoylphosphoethanolamine,
1,2-dipalmitoleylphosphoethanolamine,
1,2-dipalmitoylphosphoethanolamine,
1,2-diphytanoylphosphoethanolamine,
1,2-distearoylphosphoethanolamine,
1-palmitoyl-2-oleoylphosphoethanolamine,
1-palmitoyl-2-(10,12-tricosadinoyl)phosphoethanolamine,
1,2-dioleoylphosphoethanolamine-N-caproylamine,
1,2-dipalmitoylphosphoethanolamine-N-caproylamine,
1,2-dioleoylphosphoethanolamine-N,N-dimethyl,
1,2-dipalmitoylphosphoethanolamine-N,N-dimethyl,
1,2-dipalmitoylphosphoethanolamine-N-dodecanoyl,
1,2-dioleoylphosphoethanolamine-N-dodecanoyl,
1,2-dioleoylphosphoethanolamine-N-dodecanylamine,
1,2-dipalmitoylphosphoethanolamine-N-dodecanylamine,
1,2-dioleoylphosphoethanolamine-N-glutaryl,
1,2-dipalmitoylphosphoethanolamine-N-glutaryl,
1,2-dioleoylphosphoethanolamine-N-lactose,
1,2-dioleoylphosphoethanolamine-N-[4(p-maleimidemethyl)cyclohexan
e-carboxylate],
dipalmitolylphosphoethanolamine-N-[4(p-maleimidemethyl)cyclohexan
e-carboxylate],
1,2-dipalmitoylphosphoethanolamine-N-[4(p-maleimidephenyl)butyramide],
1,2-dioleoylphosphoethanolamine-N-[4 (p-maleimidephenyl)butyrate],
1,2-dioleoylphosphoethanolamine-N-methyl,
dipalmitoylphosphoethanolamine-N-methyl,
1,2-dioleoylphosphoethanolamine-N-[3-(2-pyridyldithio)propionate],
1,2-dipalmitoylphosphoethanolamine-N-[3-(2-pyridyldithio)propionate],
1,2-dioleoylphosphoethanolamine-N-(succinyl), and
1,2-dipalmitoylphosphoethanolamine-N-(succinyl). Of these
membrane-fusogenic lipids, dioleoylphosphatidylethanolamine is
preferably used in the carrier composition for nucleic acid
delivery of the invention.
When the carrier composition for nucleic acid delivery contains the
membrane-fusogenic lipid, the proportion of the membrane-fusogenic
lipid is not specifically limited as long as the effects of the
invention are not adversely affected. The proportion of the
membrane-fusogenic lipid is from 1 to 500 parts by weight,
preferably from 10 to 250 parts by weight, and more preferably from
25 to 100 parts by weight, based on 100 parts by weight of the
total amount of the components (A) to (C).
The carrier composition for nucleic acid delivery of the invention
can contain various additives such as isotonizing agents,
excipients, diluents, thickeners, stabilizers, buffers, and
preservatives; and aqueous vehicles such as purified water, an
aqueous saccharide solution, a buffer solution, a physiological
saline, an aqueous polymer solution, and RNase free water,
according to its form. The amounts of the additives and aqueous
vehicles can be appropriately set according to the form of the
carrier for nucleic acid delivery.
The form of carrier composition for nucleic acid delivery of the
invention is not specifically limited as long as it can include the
target nucleic acid to be delivered into cells, and the composition
is preferably in the form of a liposome.
When the carrier composition for nucleic acid delivery of the
invention is liposomal form, the components (A) to (C) and other
lipids, which are optionally added, form a liposomal membrane. When
the liposome is formed, it may be small unilamellar vesicles (SUV),
large unilamellar vesicles (LUV), or multilamellar vesicles (MLV).
Moreover, the particle diameter can be appropriately set according
to the kind of cells to be delivered, for example, the particle
diameter is 20 to 100 nm for SUV, 200 to 1,000 nm for LUV, and 400
to 3,500 nm for MLV. The particle diameter is determined using a
dynamic light scattering method.
Production of the liposome and adjustment of its particle diameter
are implemented according to methods which are common knowledge of
one skilled in the art. More specifically, the liposome can be
formed using an oil phase containing the components (A) to (C) and
a water phase (aqueous vehicle) by a thin film method, a
reverse-phase evaporation method, an ether infusion method, a
surfactant method, and a heating method. Furthermore, the particle
diameter can be adjusted by an extrusion method, a French press
method, and a homogenization method.
The carrier composition for nucleic acid delivery of the invention
is prepared by mixing the components (A) and (B) and, if necessary,
other components, and appropriately forming the mixture into a
preparation according to desired form.
Composition for Nucleic Acid Delivery
The composition for nucleic acid delivery of the invention contains
the carrier composition for nucleic acid delivery and nucleic acid.
Thus, the composition for nucleic acid delivery is used for
introducing the nucleic acid contained in the composition into
cells, which become the delivery target.
When the carrier composition for nucleic acid delivery is liposomal
form, in the composition for nucleic acid delivery, the nucleic
acid may exist in a state included in the aqueous phase of the
liposome, or a state bound to the inside or outside of a liposomal
membrane through an ionic or hydrophobic bond. In addition, if the
carrier composition for nucleic acid delivery is not liposomal
form, in the composition for nucleic acid delivery, it is only
necessary to form a complex of the nucleic acid with components of
the carrier composition for nucleic acid delivery through an ionic
or hydrophobic bond.
The composition for nucleic acid delivery of the invention is
prepared by mixing the carrier composition for nucleic acid
delivery and nucleic acid, and forming the mixture into a desired
form, or produced by mixing the nucleic acid and components of the
carrier compositions for nucleic acid delivery in any order.
In the composition for nucleic acid delivery of the present
invention, the mixing ratio of the nucleic acid and the carrier
composition for nucleic acid delivery varies depending on the kind
of nucleic acid, the carrier composition for nucleic acid delivery
used, and the kind of cells of the delivery target. The proportion
of nucleic acid is from 1.0.times.10.sup.-5 to 1.0 parts by weight,
preferably from 1.0.times.10.sup.-4 to 1.0.times.10.sup.-1 parts by
weight, and more preferably from 1.0.times.10.sup.-3 to
1.0.times.10.sup.-2 parts, per 100 parts by weight of the total
amount of the components (A) to (C) contained in the carrier
composition for nucleic acid delivery.
Also, the total amount of the components (A) to (C) contained in
the composition for nucleic acid delivery is from 10 to 90% by
weight, preferably from 30 to 80% by weight, and more preferably
from 40 to 60% by weight, based on the total amount of the
composition.
The carrier composition for nucleic acid delivery of the invention
can contain various additives such as isotonizing agents,
excipients, diluents, thickeners, stabilizers, buffering agent, and
preservatives; and aqueous vehicles such as purified water, an
aqueous saccharide solution, a buffer, a physiological saline,
according to its form. The amounts of the additives and aqueous
vehicles can be appropriately set according to the form of the
carrier for nucleic acid delivery.
In the invention, examples of the cells to which the nucleic acid
is delivered include cultured cells, cells separated from living
organisms (including established cell lines), and cells existing in
living organisms such as human.
The form of the composition for nucleic acid delivery of the
invention is not specifically limited as long as a proper amount of
the composition for nucleic acid delivery is applied so as to be
brought into contact with target cells into which the nucleic acid
is introduced.
When the nucleic acid is delivered into cells existing in living
organisms such as human, examples of the application include direct
infusion into tissue; intravenous, subcutaneous, intramuscular,
intraperitoneal, intraocular, digestive organic, and endodontic
injections; inhalation administration to the nasal cavity, mouth
cavity and lungs; oral administration; percutaneous administration
through the skin; and mucosal administration through the oral
mucous membrane, vaginal mucous membrane, ocular mucous membrane,
rectal mucous membrane, and uterine mucous membrane. Alternatively,
when the nucleic acid is delivered into cells separated from living
organisms and cultured cells, a method of culturing cells in the
presence of an appropriate amount of the composition for nucleic
acid delivery added in advance of incubation is exemplified. In
addition, when the nucleic acid is delivered into cells separated
from living organisms or cultured cells, the nucleic acid can also
be delivered into cells even in the presence of serum.
Amount of the composition for nucleic acid delivery of the
invention applied to the delivery target cells is determined
according to the kind of nucleic acid used, the kind of carrier
composition for nucleic acid delivery used, and the kind of target
cells. For example, when the delivery target are cells in human,
therapeutically effective amount of the composition for nucleic
acid delivery of the invention is administer to the patient of whom
the therapeutic gain is expected by administering the nucleic
acid.
EXAMPLES
The invention will now be described in detail based on Examples and
the like, but the invention is not limited thereto. In the
following Examples, distearoylphosphatidylcholine is abbreviated to
"DSPC", dipalmitoylphosphatidylcholine is abbreviated to "DPPC",
and dimyristoylphosphatidylcholine is abbreviated to "DMPC". In the
following Test Examples 1 and 2, GL3-siRNA (siRNA to firefly
luciferase; Dharmacon Co., Boulder, Colo., USA; sense:
5'-CUUACGCUGAGUACUUCGAdTdT, SEQ ID NO: 1, antisense:
5'-UCGAAGUACUCAGCGUAAGdTdT, SEQ ID NO: 2) was used as siRNA. In
Test Example 3, Human MMP-9-siRNA (Samchully Pharm. Co., Ltd,
Korea; Sense 5'-CCAACUAUGACCAGGAUAAdTdT-3', SEQ ID NO: 3,
antisense: 5'-UUAUCCUGGUCAUAGUUGGdTdT-3', SEQ ID NO: 4) was used as
siRNA.
Example 1
Preparation of DSPC-containing Carrier Composition for Nucleic Acid
Delivery
DSPC, cholesterol and stearylamine were weighed in a molar ratio of
7:3:1 and then dissolved in chloroform using a recovery flask. The
solution was dried under reduced pressure using a rotary evaporator
to form a lipid thin-membrane. After DEPC-treated water
(manufactured by Ambion Co.; Rnase free water) was added to the
solution in such a manner that its DSPC concentration became 30
mg/mL, the particle diameter of the resulting solution was adjusted
by passing through a membrane having a pore diameter of 100 nm
using an extruder to prepare a carrier composition for nucleic acid
delivery in a cationic liposomal form.
Example 2
Preparation of DPPC-containing Carrier Composition for Delivery of
Nucleic Acid
In the same manner as in Example 1, except that DPPC was used in
place of DSPC, a carrier composition for nucleic acid delivery in a
cationic liposomal form was prepared.
Example 3
Preparation of DMPC-containing Carrier Composition for Delivery of
Nucleic Acid
In the same manner as in Example 1, except that DMPC was used in
place of DSPC, a carrier composition for nucleic acid delivery in a
cationic liposomal form was prepared.
Example 4
Preparation of DSPC-containing Carrier Composition for Delivery of
Nucleic Acid
A solution containing siRNA in a 2 .mu.M concentration (siRNA
solution) was prepared using a solution prepared by diluting
20.times. Tris-EDTA (TE) buffer (manufactured by Invitrogen Co.) 20
times with DEPC-treated water (manufactured by Ambion Co.,
Rnasefree water). Then, an equal amount of the carrier composition
for nucleic acid delivery of Example 1 and the siRNA solution were
mixed to form a lipoplex (complex), thus obtaining a composition
for nucleic acid delivery.
Example 5
Preparation of DPPC-containing Carrier Composition for Delivery of
Nucleic Acid
A solution containing siRNA in a 2 .mu.M concentration (siRNA
solution) was prepared using a solution prepared by diluting
20.times. Tris-EDTA (TE) buffer (manufactured by Invitrogen Co.) 20
times with DEPC-treated water (manufactured by Ambion Co.,
Rnasefree water). Then, an equal amount of the carrier composition
for nucleic acid delivery of Example 2 and the siRNA solution were
mixed to form a lipoplex (complex), thus obtaining a composition
for nucleic acid delivery.
Example 6
Preparation of DMPC-containing Carrier Composition for Delivery of
Nucleic acid
A solution containing siRNA in a 2 .mu.M concentration (siRNA
solution) was prepared using a solution prepared by diluting
20.times. Tris-EDTA (TE) buffer (manufactured by Invitrogen Co.) 20
times with DEPC-treated water (manufactured by Ambion Co.,
Rnasefree water). Then, an equal amount of the carrier composition
for nucleic acid delivery of Example 3 and the siRNA solution were
mixed to form a lipoplex (complex), thus obtaining a composition
for nucleic acid delivery.
Test Example 1
Test for Evaluation of Safety for Cells
The evaluation was performed using a MTS assay. A CellTiter 96
Aqueous One Solution Cell Proliferation Assay manufactured by
Promega Co. was used for the MTS assay. Specifically, A594 cells
(ATCC, USA) were inoculated at 3.16.times.10.sup.4 cells/well into
200 .mu.l of Dulbecco's Modification of Eagle's Medium (DMEM)
containing 10 vol % fetal bovine serum (FBS) in a 96-well plate,
and incubated at 37.degree. C. for 24 hours. After rinsing with
Hank's Buffered Salt Solution (HBSS) 3 times, the medium was
changed to DMEM without FBS, then 20 .mu.l of each of the
compositions for nucleic acid delivery of Examples 4 to 6 were
added to each well and incubated at 37.degree. C. under 5% CO.sub.2
for 4 hours. Next, the culture supernatant in the wells was changed
to DMEM with 10 vol % of FBS and incubated at 37.degree. C. under
5% CO.sub.2 for 20 hours again. 20 .mu.l of a MTS
(Methanethiosulfonate) reagent and 100 .mu.l of a DMEM medium with
10 vol % FBS were added to each well, incubated for 2 hours,
followed by determination of absorbance at 492 nm and further
calculation of cell viability. The cell viability was calculated by
setting the absorbance at 492 nm determined without adding the
composition for nucleic acid delivery which was incubated under the
above conditions as 100%.
The results are shown in FIG. 1. As shown in FIG. 1, it became
apparent that all compositions for nucleic acid delivery of
Examples 4 to 6 have low cytotoxicity and high safety.
Particularly, it was confirmed that safety was significantly high
in the compositions for nucleic acid delivery using DSPC or DPPC as
the diacylphosphatidylcholine (Example 4 or 5).
Test Example 2
Test for Evaluation of siRNA Delivery Efficiency into Cells
Intracellular introduction of siRNA was evaluated by measuring
fluorescence intensity of FITC-labeled siRNA using flow cytometry.
In this test, a composition for nucleic acid delivery prepared with
FITC pre-labeled siRNA was used. Specifically, A594 cells (ATCC,
USA) were inoculated at 5.times.10.sup.5 cells/well into 500 .mu.l
of DMEM with 10 vol % FBS in a 24-well plate, and incubated at
37.degree. C. under 5% CO.sub.2 for 24 hours. After rinsing three
times with HBSS, the medium was changed to DMEM containing no FBS,
and then 0.05 ml of each of the compositions for nucleic acid
delivery of Examples 4 to 6 were added to each well and incubated
at 37.degree. C. under 5% CO.sub.2 for 4 hours. Next, the culture
supernatant in the wells was changed to DMEM with 10 vol % of FBS
and incubated at 37.degree. C. under 5% CO.sub.2 for 20 hours
again. Each well was rinsed with HBSS once and 0.2 mL of
CellScrubBuffer (Gene Therapy Systems, Inc.) was added, followed by
incubation at 37.degree. C. under 5% CO.sub.2 for 15 minutes.
Again, the wells were rinsed with HBSS 2 times and cells attached
on the well bottom were detached using trypsin and collected by
centrifugation, and then the resulting cells were suspended in
HBSS. The suspension was filtered through a membrane having a pore
diameter of 41 .mu.m. Fluorescence intensity of cells was measured
using flow cytometry at 2 hours and 24 hours after the addition of
the compositions for nucleic acid delivery. As a control,
fluorescence intensity of a control composition for nucleic acid
delivery obtained by mixing a solution of Lipofectamine 2000.TM.
(Invitrogen) often used as a commercially-available gene vector
diluted with OptiMEM media to 0.1 mg/mL and siRNA solution diluted
with TE buffer at a concentration of 2 .mu.M in a volume ratio of
1:1 was also measured in the same manner as described above.
The results are shown in FIG. 2. From these results, it was
confirmed that siRNA is incorporated into cells in any case of
compositions for nucleic acid delivery of Examples 4 to 6.
Particularly, in the compositions for nucleic acid delivery using
DSPC or DMPC as the diacylphosphatidylcholine (Examples 4 and 6),
it became apparent that introduction of siRNA 2 hours after
addition was significantly high as compared with Lipofectamine 2000
often used as a commercially-available gene vector and that the
compositions have favorable characteristics in excellent
fast-acting.
Furthermore, a carrier for nucleic acid delivery having a DSPC
concentration of 7.5 to 30 mg/mL was produced according to the same
manner as in Example 1, using DSPC, cholesterol, and stearylamine
(DSPC:cholesterol:stearylamine=7:3:1 in a molar ratio, hereinafter,
collectively referred to as a "constituent lipid of the carrier for
nucleic acid delivery") . A lipoplex (complex) was formed by mixing
an equal amount of this carrier for nucleic acid delivery and TE
buffer containing 200 nM siRNA, and the composition for nucleic
acid delivery were prepared. Using the composition for nucleic acid
delivery prepared in this manner, introduction of siRNA into cells
was evaluated in the same manner as described above. The results
are shown in FIG. 3. From these results, when the concentration of
the constituent lipid of the carrier for nucleic acid delivery was
changed, the amount of nucleic acid introduced into cells mediated
by the composition for nucleic acid delivery was changed
accordingly. Also, it was found that the constituent lipid of the
carrier for nucleic acid delivery shows a low value in the
introduced siRNA amount at 24 hours after addition as compared with
2 hours after addition at any concentration, and that disappearance
of siRNA started at 24 hours after addition. Therefore, the results
also revealed that it is possible to deliver siRNA into cells
within a short time of 2 hours after addition by using the
combination of DSPC, cholesterol, and stearylamine as the carrier
for nucleic acid delivery, and that the compositions have favorable
characteristics in excellent fast-acting.
Test Example 3
Test for Evaluation of Inhibition of Interferon Induction
A594 cells (ATCC, USA) were inoculated at 5.times.10.sup.5
cells/well into 500 .mu.l of DMEM with 10 vol % FBS in a 24-well
plate, and incubated at 37.degree. C. under 5% CO.sub.2 for 24
hours. After rinsing three times with HBSS, a 450 .mu.l of DMEM
medium containing no FBS was added into each well, moreover, 50
.mu.l of the composition for nucleic acid delivery of Example 4 was
added into each well and incubated at 37.degree. C. under 5%
CO.sub.2 for 4 hours. Next, the culture supernatant in the wells
was changed to DMEM with 10 vol % of FBS and incubated at
37.degree. C. under 5% CO.sub.2 for 20 hours again. After rinsing
three times with HESS, cells attached on the well bottom were
detached using trypsin and collected by centrifugation. RNA was
extracted from the resulting cells using Rneasy Plus Mini (Qiagen),
and then cDNA was obtained by transcription using QutantiTect
Reverse Transcription (Qiagen). Using the resulting cDNA,
QutantiTectPrimer Assay (Qiagen), and iCycler iQ (Bio-RAD), mRNA of
IFIT-1, which is an interferon-inducing gene, was quantified with
real-time PCR. As a control, mRNA of IFIT-1 was quantified in a
control composition for nucleic acid delivery obtained by mixing a
solution of Lipofectamine 2000.TM. (Invitrogen) often used as a
commercially-available gene vector diluted with OptiMEM media to
0.1 mg/mL and the siRNA solution diluted with TE buffer at
concentration of 2 .mu.M in a volume ratio of 1:1 in the same
manner as described above. As a housekeeping gene to correct the
quantification, 18rRNA was used. Also, as a blank, mRNA of IFIT-1
was quantified with the above conditions without the addition of
the composition for nucleic acid delivery.
The results are shown in FIG. 4. The composition for nucleic acid
delivery of Example 4 using DSPC indicated significantly low
induction of interferon as compared with the control composition
for nucleic acid delivery using Lipofect.TM. amine 2000. The
results revealed that it is possible to inhibit induction of
interferon, which is an adverse effect of siRNA, using the
composition for nucleic acid delivery of the present invention.
SEQUENCE LISTINGS
1
4121DNAartificialGL3-siRNA 1cuuacgcuga guacuucgat t
21221DNAartificialGL3-siRNA 2ucgaaguacu cagcguaagt t
21321DNAartificialMMP-9-siRNA 3ccaacuauga ccaggauaat t
21421DNAartificialMMP-9-siRNA 4uuauccuggu cauaguuggt t 21
* * * * *
References